Protective effects of beet leaf extract (Beta vulgaris) on lipid profile and oxidative stress in HepG2 and HUVEC cells

Orientadores: Fernando Moreira Simabuco, Rosângela Maria Neves BezerraDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências AplicadasResumo: A beterraba é uma herbácia com colo tuberizado muito utilizada pelo mundo, não só como um produto alimentício, mas também como matéria prima para outros setores, como o setor de produção alcoólica, de açúcar e fonte de corante alimenticío. Suas folhas, entretanto, são produtos alimentícios não convencionais no Brasil (PANCs), e possuem propriedades funcionais parecidas com as do espinafre as quais já foram comprovadas. Estudos do grupo do Laboratório Multidisciplinar em Alimentos e Saúde (LABMAS) mostraram que o consumo diário de folhas de beterraba é vantajoso para pacientes obesos dislipidêmicos, já que seu consumo melhora parâmetros colesterolêmicos e insulinêmicos. Esse trabalho objetivou complementar os resultados da melhora no perfil de LDL na presença do extrato das folhas da beterraba através da elucidação das vias metabólicas nas quais o mesmo atua, usando modelos de cultura de células endoteliais e hepatócitos, assim como técnicas de biologia celular. O extrato das folhas da beterraba mostrou, em modelos in vitro, melhorar a proteção oxidativa em células endoteliais de vasos sanguíneos humanos (HUVEC) contra diferentes agentes incluindo LDL-ox e H2O2. O extrato se mostrou eficaz também, na taxa de adesão de monócitos à monocamada de células endoteliais, passo precursor da formação do ateroscleroma. Além disso, o extrato de folhas de beterraba foi capaz de alterar a biossíntese do colesterol endógeno nas células de fígado (HEPG2), modulando a expressão gênica de proteínas cruciais para a síntese do mesmo, como HMG-CoA e a apolipoproteína B, agindo também na inibição da oxidação do LDL e regulando lipídeos intracelulares, se mostrando um potencial nutracêutico. Por fim, as folhas da beterraba mostraram agir não somente na via colesterol intracelular, mas também, diretamente na proteção oxidativa gerada pelo LDL em sua forma oxidada e outras fontes de estresse oxidativo, como o H2O2. Embora os resultados mostrem que tais efeitos não se dão completamente devido aos derivados de apigenina, é evidente o benefício do consumo sinérgico de todos os compostos presentes no extrato da folha de beterraba.Abstract: Beetroot is a tuberized cervical herb widely used around the world, not only as a food, but also as a raw material for other sectors such as the alcoholic, sugar and food coloring sector. Its leaves, however, are unconventional food in Brazil (PANCs), and have functional properties similar to those of spinach which have already been proven. Studies from the Multidisciplinary Food and Health Laboratory (LABMAS) group have shown that daily consumption of beet leaves is advantageous for dyslipidemic obese patients, seen that their consumption improves cholesterolemic and insulinemic parameters. This study aimed to complement the results of the improvement in LDL profile in the presence of beet leaf extract by elucidating the metabolic pathways in which it acts, using endothelial cell and hepatocyte culture models, as well as cell biology techniques. Beet leaf extract has been shown in in vitro models to improve oxidative protection in human blood vessel endothelial cells (HUVEC) against different agents including LDL-ox and H2O2. The extract was also effective in the rate of monocyte adhesion to the endothelial cell monolayer, a precursor step of atheroscleroma formation. In addition, beet leaf extract was able to alter endogenous cholesterol biosynthesis in liver cells (HEPG2) by modulating gene expression of proteins crucial for its synthesis, such as HMG-CoA and apolipoprotein B, also acting to inhibit oxidation of LDL and regulating intracellular lipids, showing a nutraceutical potential. Finally, sugar beet leaves have been shown to act not only on the intracellular cholesterol pathway, but also on the oxidative protection generated by LDL in its oxidized form and other sources of oxidative stress, such as H2O2. Although the results show that such effects are not completely due to apigenin derivatives, the benefit of the synergistic consumption of all the compounds present in the beet leaf extract is evident.MestradoCiências Nutricionais e MetabolismoMestre em Ciências da Nutrição e do Esporte e Metabolismo88882.435835/2019-01CAPE

Survival, metabolic status and cellular morphology of probiotics in dairy products and dietary supplement after simulated digestion

The objective of the present study was to compare the effect of dairy matrices (fermented milk and ice cream) and a dietary supplement containing the same amounts of inulin and the same viable counts of Lactobacillus acidophilus and Bifidobacterium animalis on the survival of probiotics when submitted to simulated digestion. The survival was evaluated by plate counts in MRS agar, the metabolic status by flow cytometry and the cell morphology by scanning electronic microscopy. The percentages of live cells observed by flow cytometry and by plate counts were higher in the fermented milk when compared with the other products (p <0.05). The cell integrity was better maintained in the dairy products as compared to the dietary supplement. The dairy matrices were more effective in maintaining the viability of probiotics during the digestion process than the dietary supplement, and the fermented milk proved to be the best way of delivering viable probiotics

Protective effects of beet (beta vulgaris) leaves extract against oxidative stress in endothelial cells in vitro

Beetroot is an herb used worldwide as a food product, raw material for food industry, ethanol production and source of food coloring. Beet leaves are an unconventional food with antioxidant properties, which might neutralize reactive oxygen species (ROS) induced by oxidized Low-Density Lipoprotein (LDL) present in dyslipidemias. This study aimed to elucidate the effects of beet leaves on the suppression of LDL oxidative processes. Beet leaves extract was produced, characterized, and tested for its antioxidant capacity using endothelial cells in vitro. A model of human umbilical vein endothelial cells was used in various tests, including viability assay, molecular analysis of antioxidant genes, ROS labeling, and macrophage adhesion assay. The extract improved the antioxidative protection of endothelial cells against different agents including oxidized LDL-cholesterol and H2O2. It acted on ROS directly due to its high content of natural antioxidants, but also due to the activation and improvement of cellular defenses such as Superoxide dismutase 1, Superoxide dismutase 2, and catalase. The inhibition of LDL-mediated oxidative effects on endothelial cells may turn this unconventional food a functional food with great potential for phytotherapy of atherosclerosis as an adjuvant for medicinal treatmentsCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo303568/2016‐0; 447553/2014‐32012/13558‐7; 2013/04304‐4; 2016/06457‐0; 2018/14818‐

Pomegranate juice and peel extracts are able to inhibit proliferation, migration and colony formation of prostate cancer cell lines and modulate the akt/mtor/s6k signaling pathway

Pomegranate (Punica granatum) is known to contain polyphenols with many potential health benefits, including anti-tumoral, anti-inflammatory, and anti-microbial properties. It has been used in popular medicine for cancer treatment, which still represents the major cause of cancer-related deaths in men worldwide. Importantly, pomegranate peels are valuable by-products of the food industry that are rich in polyphenols. Here we report a comparison between juice and peel aqueous extracts in prostate cancer DU-145 and PC-3 cell lines. Both extracts were able to inhibit the proliferation, migration and colony formation of those cells, although peel extracts presented more robust effects compared to juice. Besides, the growth-related mTOR/S6K signaling pathway presented strong inhibition after pomegranate extracts treatment. This study presents evidence that both juice and isolated peel extracts from promegate fruit have important anti-cancer effects against prostate cancer cells, modulating the mTOR/S6K signaling pathway755462CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP447553/2014-3; 303568/2016-02012/13558-7;2018/14818-9; 2015/07299-7; 2016/06457-0; 2013/04304-4; 2015/003111; 2013/13002-

NEK6 Regulates Redox Balance and DNA Damage Response in DU-145 Prostate Cancer Cells

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy

Short-term high-fat diet modulates several inflammatory, ER stress, and apoptosis markers in the hippocampus of young mice

The consumption of saturated fatty acids is one of the leading risk factors for Alzheimer's Disease (AD) development. Indeed, the short-term consumption of a high-fat diet (HFD) is related to increased inflammatory signals in the hippocampus; however, the potential molecular mechanisms linking it to AD pathogenesis are not fully elucidated. In our study, we investigated the effects of short-term HFD feeding (within 3, 7 and 10 days) in AD markers and neuroinflammation in the hippocampus of mice. The short period of HFD increased fasting glucose and HOMA-IR. Also, mice fed HFD increased the protein content of beta-Amyloid, pTau, TNF alpha, IL1 beta, pJNK, PTP1B, peIF2 alpha, CHOP, Caspase3, Cleaved-Caspase3 and Alzheimer-related genes (Bax, PS1, PEN2, Aph1b). At 10 days, both neuronal (N2a) and microglial (BV2) cells presented higher expression of inflammatory and apoptotic genes when stimulated with palmitate. These findings suggest that a short period of consumption of a diet rich in saturated fat is associated with activation of inflammatory, ER stress and apoptotic signals in the hippocampus of young mice79284293COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP306535/2017-3; 309339/2016-2; 462410/2014-52016/18488-8Obesity and Comorbidities Research Center - OCRC; CNPqNational Council for Scientific and Technological Development (CNPq) [306535/2017-3, 309339/2016-2, 462410/2014-5]; FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2016/18488-8

Increased mTOR Signaling and Impaired Autophagic Flux Are Hallmarks of SARS-CoV-2 Infection

The COVID-19 (Coronavirus Disease 2019), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), severely affects mainly individuals with pre-existing comorbidities. Here our aim was to correlate the mTOR (mammalian/mechanistic Target of Rapamycin) and autophagy pathways with the disease severity. Through western blotting and RNA analysis, we found increased mTOR signaling and suppression of genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2 as well as in transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients. Immunofluorescence co-localization assays also indicated that SARS-CoV-2 colocalizes within autophagosomes but not with a lysosomal marker. Our findings indicate that SARS-CoV-2 can benefit from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling